A complete list of references can be found at the end of the specification.
Cellulose is one of the most abundant polymers found in nature and consists of glucose units connected by beta 1,4 linkages. The beta 1,4 linkages which connect individual glucose units are not easily degraded or depolymerized. However, there exists a variety of cellulase enzymes which are capable of enzymatically hydrolyzing cellulose.
Cellulases are enzymes produced by a number of microorganisms which catalyse the hydrolysis of cellulose to products such as glucose, cellobiose, and other cellooligosaccharides. Cellulase is usually a generic term denoting a multienzyme mixture comprising exo-cellobiohydrolases (CBH), endoglucanases (EG) and β-glucosidases. Cellulase produced by the filamentous fingi Trichoderma longibrachiatum comprises at least two cellobiohydrolase enzymes termed CBHI and CBBE and at least 4 EG enzymes.
Cellulase enzymes work synergistically to degrade cellulose to glucose. CBHI and CBHII generally act on the ends of the glucose polymers in cellulose microfibrils liberating cellobiose (Teeri and Koivula, 1995) while the endoglucanases act at random locations on the cellulose. Together these enzymes hydrolyse cellulose to smaller cello-oligosaccharides such as cellobiose. Cellobiose is hydrolysed to glucose by β-glucosidase.
The genes encoding CBHI, CBH II (Shoemaker et al., 1983; Teeri et al., 1987), EG I and EG II (Penttila et al., 1986; Saloheimo et al., 1988) have been cloned and isolated from filamentous fungi such as T. reesei and T. longibrachiatum. CBHI, CBH II and most of the major EG enzymes comprise a catalytic core domain and a cellulose binding domain (CBD) separated by a flexible linker region. The cellulose binding domain (CBD) promotes adsorption of the enzyme to regions of the cellulosic substrate (Tomme et al., 1988; Gilkes et al, 1992), while the core domain is responsible for catalysing the cleavage of cellulose. The linker region may ensure an optimal interdomain distance between the core domain and the cellulose binding domain (Teeri et al., 1992).
Proteins consisting of either the isolated CBD or the core protein have been produced and studied. Core proteins of CBHI are also found in amounts of less than about 10 % in cellulase mixtures obtained from natural sources. Studies on the isolated fungal catalytic domain (core protein) suggest that this protein is capable of binding to cellulose although with reduced affinity compared to the native Polo) protein (Tomme et al., 1988). The strong binding imparted to cellulases by its CBD suggests that the adsorption of several cellulases to cellulose is essentially irreversible (Beldman et al., 1987; Kyriacou et al., 1989)
CBHI core protein from Trichoderma reesei does not bind as tightly to cellulose as CBHI. The CBHI core protein is fully active against small soluble substrates such as the chromophoric glycosides derived from the cellodextrins and lactose. However, its activity against an insoluble cellulosic substrates such as Avicel (a crystalline type of cellulose) is greatly reduced compared to CBHI (Van Tilbeurgh et al., 1986). Van Tilbeurgh showed that CBHI-core was less than 1% as active as CBHI. This was attributed to the fact that 88% of the CBHI adsorbed to the cellulose versus only 36% of the CBHI-core protein. Kim et al., (1997) examined the absorption and activities of CBHI, CBHI core and other protein mixtures on Avicel. They disclose that CBMI core produced less than ¼ the amount of reducing sugar from Avicel than CBHI. The higher rate of hydrolysis by CBHI was attributed to its better binding. In similar experiments with steam-pretreated willow, Kotiranta et al. (1999) observed a drastically reduced hydrolysis rate for CBHI core compared to intact CBHI and concluded that CBHI needs a CBD for efficient adsorption and hydrolysis. Nidetsky et al. (1994) observed similar trends between CBHI-core and CBHI. Over 80% of the CBHI adsorbed to cellulose filter paper, compared with only 40% of CBHI-core. The rate of hydrolysis of the core and CBHI were directly proportional to the amount of adsorbed protein, with CBHI being more than twice as active as CBHI-core. These studies indicate that there is no advantage in using CBHI-core rather than CBHI for cellulose hydrolysis, since the activity of CBHI-core against crystalline cellulose is much slower than CBHI.
The conversion of cellulose from cellulosic material into glucose is important in many industrial processes, such as the bioconversion of cellulose to fuel ethanol. Unfortunately, cellulose contained in most plant matter is not readily convertible to glucose, and this step represents a major hurdle in the commercialization of such a process. The efficient conversion of cellulose from cellulosic material into glucose was originally thought to involve liberating cellulose and hemicellulose from their complex with lignin. However, more recent processes focus on increasing the accessibility to cellulose within the lignocellulosic biomass followed by depolymerization of cellulose carbohydrate polymers to glucose. Increasing the accessibility to cellulose is most often accomplished by pretreating the cellulosic substrate.
The goal of most pretreatment methods is to deliver a sufficient combination of mechanical and chemical action, so as to disrupt the fiber structure and improve the accessibility of the feedstock to cellulase enzymes. Mechanical action typically includes the use of pressure, grinding, milling, agitation, shredding, compression/expansion, or other types of mechanical action. Chemical action typically includes the use of heat (often steam), acid, and solvents. For example, one of the leading approaches to pretreatment is by steam explosion, using the process conditions described in (U.S. Pat. No. 4,461,648) and also in Foody et al., 1980), both of which are incorporated herein by reference). In this process, lignocellulosic biomass is loaded into a steam gun and up to 5% acid is optionally added to the biomass in the steam gun or in a presoak prior to loading the steam gun. The steam gun is then filled very quickly with steam and held at high pressure for a set length of cooking time. Once the cooking time elapses, the vessel is depressurized rapidly to expel the pretreated biomass.
Another approach described in U.S. Pat. No. 4,237,226, discloses the pretreatment of oak, newsprint, poplar, and corn stover by a continuous plug-flow reactor, a device that is similar to an extruder. Rotating screws convey a feedstock slurry through a small orifice, where mechanical and chemical action break down the fibers.
Pretreatment has been suggested to enhance delignification of the cellulosic substrate (Fan et al., 1981), create micropores by the removal of the hemicellulose, change the crystallinity of the substrate, and reduce the degree of polymerization of the cellulose (Knappert et al., 1980) and increase the surface area of the cellulosic substrate (Grethlein and Converse, 1991; Grohman et al., 1985).
Unfortunately, to date the approach of a pretreatment coupled with enzyme hydrolysis has not been able to produce glucose at a sufficiently low cost, so as to make the conversion of cellulose to ethanol commercially attractive. Even with the most efficient of the currently known pretreatment processes, the amount of cellulase enzyme required to convert cellulose to glucose is high and this represents a significant cost in ethanol production. The option of adding less cellulase to the system usually decreases the amount of glucose produced to an unacceptable extent. The approach of decreasing the amount of enzyme required by increasing the length of time that the enzyme acts on the cellulose leads to uneconomical process productivity, stemming from the high cost associated with retaining the enzymatic mixtures in hydrolysis tanks.
Thus there is a need within the art to identify new methods which enhance the conversion of cellulose within a cellulosic substrate to glucose. Further there is a need in the art to identify enzymes or mixtures of enzymes which enhance the conversion of cellulose to glucose and which are recoverable, recyclable, and reusable.
It is an object of the present invention to overcome drawbacks of the prior art.
The above object is met by a combination of the features of the main claims. The sub claims disclose further advantageous embodiments of the invention.